Method of treating tumors

ABSTRACT

In accordance with the present invention, a method is provided for treating tumors. More particularly, the present invention relates to a method for treating tumors comprising injecting a solution of certain ethylene oxide-propylene oxide surface-active copolymers and a solution containing chemotherapeutic agents into a human or animal with a tumor. The solutions can be injected simultaneously or separately at different times. In addition, the surface-active copolymer and the chemotherapeutic agent can be in the same solution. The surface-active copolymer can be an ethylene oxide-propylene oxide condensation product with the following general formula: 
     
         HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (O.sub.2 H.sub.4 
    
      O) b  H 
     wherein a is an integer such that the hydrophobe represented by (C 3  H 6  O) has a molecular weight of approximately 950 to 4000 daltons, preferably approximately 1200 to 3500 daltons, and b is an integer such that the hydrophile portion represented by (C 2  H 4  O) constitutes approximately 50% to 90% by weight of the compound.

This is a continuation of U.S. patent application Ser. No. 08/259,147,filed Jun. 13, 1994, now abandoned, which is a continuation of U.S.patent application Ser. No. 08/131,865, filed Oct. 5, 1993, nowabandoned, which is a continuation of U.S. patent application Ser. No.07/881,203, filed May 11, 1992, now abandoned, which is a continuationof U.S. patent application Ser. No. 07/802,331, filed on Dec. 4, 1991,now U.S. Pat. No. 5,198,211, which is a continuation of U.S. patentapplication Ser. No. 07/522,206, filed on May 11, 1990, now U.S. Pat.No. 5,078,955, which is a continuation of U.S. patent application Ser.No. 07/403,017, filed on Sep. 5, 1989, now abandoned, which is acontinuation of U.S. patent application Ser. No. 07/303,791, filed onJan. 30, 1989, now abandoned, which is a division of U.S. patentapplication Ser. No. 07/045,459, filed on May 7, 1987, now U.S. Pat. No.4,801,452, which is a continuation of U.S. patent application Ser. No.07/043,888, filed on Apr. 29, 1987, now abandoned, which is acontinuation of U.S. patent application Ser. No. 06/863,582, filed onMay 15, 1986, now abandoned.

TECHNICAL FIELD

The present invention relates to a composition and method for lysingfibrin clots, dissolving thrombi, and reestablishing and maintainingperfusion of ischemic tissue. More particularly, the present inventionrelates to a composition comprising certain ethylene oxide-propyleneoxide condensation products, which are surface-active copolymers, incombination with fibrinolytic enzymes.

BACKGROUND OF THE INVENTION

The term "fibrinolytic enzyme" means any enzyme that is capable ofcleaving fibrin. Enzymes that are capable of cleaving fibrin include,but are not limited to, streptokinase, urokinase, tissue plasminogenactivator (t-PA) produced from cell cultures, tissue plasminogenactivator produced by recombinant DNA technology (rt-PA) and tissueplasminogen activator produced by prourokinase (k-PA). The terms"isotonic solution" or "isoosmotic solution" are defined as solutionshaving the same or similar osmotic pressure as blood. The terms clot,fibrin clot and thrombus are used interchangeably.

Each year about 550,000 Americans die from heart attacks. Evenmore--close to 700,000--have heart attacks and live. While a hem attackvictim may survive, pan of his or her heart will almost certainly die.The death of heart muscle, called myocardial infarction, is due tocoronary artery thrombosis in 70-90% of the cases. When a thrombosis, orblood clot, occludes one of the arteries of the heart, it compromisesthe flow of blood to the surrounding muscle This deprives the muscle ofoxygen and other nutrients. In the past, nothing could be done toreverse this process. The high technology devices in intensive careunits mostly supported patients so they could live while a portion oftheir heart died.

Similar situations occur in many other tissues when the blood supply tothe tissue is affected by a thrombus or embolus. Stroke, deep veinthrombosis and pulmonary embolus are examples.

Another area where fibrinogen/fibrin plays a role is tumors. There isnow strong evidence that fibrinogen-related proteins are localized insolid tumors. The anatomical distribution of fibrin in tumors variesdepending on the tumor type. In carcinomas, fibrin is deposited in thetumor stroma and around tumor nests and may be particularly abundanttoward the tumor periphery and at the tumor host interface. By contrast,fibrin is often less prominent in older, more central tumor stromacharacterized by sclerotic collagen deposits. Fibrin may also be foundbetween individual carcinoma cells. In some, but not all such cases,interepithelial fibrin deposits are related to zones of tumor necrosis;however, zones of minor necrosis are not necessarily sites of fibrindeposition. Fibrin deposition in sarcomas has been less carefullystudied than that in carcinomas. In lymphomas, fibrin deposits may beobserved between individual malignant tumor cells as well as betweenadjacent, apparently reactive benign lymphoid elements. Fibrin has beenreported to appear in zones of tumor sclerosis, as in Hodgkin's disease.Research has indicated that the pattern and extent of fibrin depositionare characteristic for a given tumor. (See Hemostasis and Thrombosis,Basic Principles and Clinical Practice, "Abnormalities of Hemostasis inMalignahoy", pp. 1145-1157, ed. by R. W. Colman, et al., J. B.Lippincott Company, 1987).

The lack of a uniform vascular supply to tumors can impede diagnosticand therapeutic procedures. For example, hypoxic minors are lesssusceptible to many drags and to radiation. Conventional drugs and newdrugs, such as monoclonal antibody conjugates, are not effective unlessthey are delivered to tumor cells. Fibrin deposits that surround sometypes of tumors inhibit delivery of the drugs to the minor. The bloodsupply of tumors is further compromised by other factors as well. Bloodvessels in tumors are frequently small and tortuous. The hydrodynamicresistance of such channels further impedes the flow of blood to tumors.

A similar situation occurs for different reasons during crisis of sicklecell anemia. Sickled red blood cells partially occlude small vesselsproducing local hypoxia and acidosis. This induces additional red bloodcells to become sickled. The result is a vicious circle known as"crisis". Therapy involves increasing flow and oxygenation in affectedareas. Another therapy involves the combination of calcium channelblockers. The formation of fibrin frequently complicates sickle cellcrisis.

It has been found that certain enzymes are able act on fibrin depositsto open clogged arteries. The enzymes which have been used successfullyinclude streptokinase, urokinase, prourokinase, tissue plasminogenactivator produced from cell cultures and tissue plasminogen activatorproduced by recombinant DNA technology. These enzymes are mostsuccessful if administered shortly after the occlusion of the bloodvessels before the heart tissue has sustained irreversible damage. Inone study of 11,806 patients treated with intravenous or intracoronaryartery streptokinase, an 18% improvement of survival was demonstrated.If the treatment was begun within one hour after the initial pain onsetof the heart attack, the in-hospital mortality was reduced by 47%. (SeeThe Lancet, Vol. 8478, p. 397-401, Feb. 22, 1986). It was demonstratedthat early lysis of the thrombus resulted in salvage of a portion ofheart tissue which would otherwise have died. In studies usingangiography to assess the patency of blood vessels, it was found thattissue plasminogen activator could completely open the vessels of 61% ofthe 129 patients versus 29% of controls who were not treated with theenzyme. (See Verstraete, et al., The Lancet, Vol. 8462, p. 965-969, Nov.2, 1985). Tissue plasminogen activator requires the addition ofapproximately 100 μl of Tween 80 per liter of solution to promotedispersion of the enzyme. (See Korninger, et al., Thrombos, Haemostas,(Stuttgart) Vol. 46(2), p. 561-565 (1981)).

The enzymes used to lyse thrombi in vessels do so by activatingfibrinolysis. Fibrin is the protein produced by polymerization offibrinogen. It forms a gel which holds the thrombus together. The fibrinmolecules which form clots gradually become cross-linked to make a morestable clot. All four enzymes, prourokinase, urokinase, streptokinaseand tissue plasminogen activator, have similar effects on fibrin;however, they have different toxicities. If the fibrinolysis mechanismsare activated in the vicinity of a clot, the clot is lysed. If, however,they am activated systemically throughout the circulation, the body'scapacity to stop bleeding or hemorrhage is markedly reduced.Streptokinase and urokinase tend to activate systemic fibrinolysis.Consequently, they have been most effective when injected directly intothe affected blood vessel. Tissue plasminogen activator, in contrast,becomes effective only when it is actually attached to fibrin. Thismeans its activity is largely localized to the immediate area of a clotand does not produce systemic fibrinolysis. If high doses are used in aneffort to increase the rate of clot lysis or to lyse refractory clots,then the mount of systemic fibrinolysis and risk of hemorrhage becomesignificant. It can be injected intravenously into the generalcirculation. It circulates harmlessly until it contacts the fibrin in ablood clot where it becomes activated and lyses the clot. Tissueplasminogen activator is able to lyse a clot which is extensivelycross-linked. This means it is possible to lyse clots which have beenpresent for many hours. Tissue plasminogen activator also produces lessrisk of hemorrhage than the other enzymes. Even more effective enzymebased thrombolytic drugs are being developed.

Remarkable as the new enzyme therapies are, they are subject to seriouscomplications and are not effective in all patients. Clots in theanterior descending branch of the left coronary artery are much morereadily lysed than those in other arteries. If the enzyme is notdelivered by the blood stream directly to the thrombus, it has noeffect. For various reasons, more blood passes by or trickles aroundthrombi in the left anterior descending coronary artery than in theother major arteries. In addition, the presence of collateralcirculation which forms in response to compromised blood flow in themajor arteries adversely affects the rate of reopening or recanalizationof the thrombosed major arteries. It is thought the presence of manycollateral vessels which allows blood to bypass the clot reduces thepressure gradient across the clot. This in turn reduces the blood flowthrough the tiny openings which may persist in the clot, impedes thedelivery of enzymes to the clot, and prevents it from being lysed.

Once the clot is lysed, the factors which led to the formation of thethrombus persist. This produces a high incidence of re-thrombosis andfurther infarction in the hours and days following lysis of the clot.Rethrombosis has been reported in between 3% and 30% of cases in whichthe initial treatment successfully lysed the clot. Anticoagulants arecurrently used to prevent the formation of new thrombi, but they tend toinduce hemorrhage. There is a delicate balance between the amount ofanticoagulation necessary to prevent re-thrombosis of the vessels andthat which will produce serious hemorrhage.

Finally, dissolving the clot after irreversible damage has taken placehas little effect. The irreversible damage could be either to the heartmuscle or vascular bed of the tissue supplied by the blood vessel. Amajor problem in widespread implementation of this new enzyme therapy isto find ways of identifying and treating the patients earlier in theirdisease and to find ways to make the treatment effective for a longerperiod of time after the initiation of thrombosis.

Animal studies have provided a better understanding of the events whichcontrol blood flow and tissue death following coronary arterythrombosis. The heart has several blood vessels, so much of the musclereceives blood from more than one vessel. For this and other reasons,the tissue changes following a coronary thrombosis are divided intodistinct zones. The central zone of tissue becomes almost completelynecrotic. This is surrounded by an area of severe ischemia. Outside thisis an area of lesser ischemia called the marginal zone. Finally, thereis a jeopardized zone which surrounds the entire area. In studies withbaboons, the central necrotic area was not affected by recanalization ofthe vessel after several hours. However, muscle in the other zones whichhad undergone less severe damage during the ischemic period could besalvaged. A surprising finding was that lysing of the thrombus toproduce a perfect arteriograph was insufficient to restore normal flowin the majority of animals. (See Flameng, et al, J. Clin. Invest., Vol.75, p. 84-90, 1985).

Some further impediment to flow had developed in the area supplied bythe vessel during the time that it was occluded. In further studies, itwas demonstrated that immediately after removing the obstruction to thevessel, the flow through the damaged tissue began at a high rate.However, within a short time the blood flow through the ischemic zonedecreased and the tissue died. Consequently, the regional blood flowimmediately after reperfusion is a poor predictor of the salvage ofmyocardial tissue. If the blood flow through the damaged tissue remainednear the normal levels, the success of tissue salvage was much greater.Hemorrhage occurred almost exclusively in the severely ischemic zonereflecting damage to the small blood vessels. The hemorrhage, however,remained limited to the severely ischemic tissue and did not causeextension of the infarction or other serious complication. Therapieswhich could preserve the blood flow through the small blood vesselsdistal to the major area of thrombus after reperfusion could be expectedto markedly increase the salvage of myocardial tissue.

The damage to heart muscle cells which occurs after lysing the thrombusis due to other factors as well as ischemia. Contact of fresh blood withdamaged or dead cells induces the influx of neutrophils, or pus cells,which kill heart cells which would otherwise have recovered. Much of thedamage caused by neutrophils has been attributed to superoxide ions. Thesuperoxide anion can damage tissue in several ways. The interaction ofthe superoxide anion with hydrogen peroxide leads to the production ofhydroxyl radicals which are potentially toxic and react rapidly withmost organic molecules. Mannitol is a selective scavenger of hydroxylradicals. The enzyme, superoxide dismutase, catalyzes the decompositionof the superoxide anion. Enzymes such as superoxide dismutase, freeradical scavengers or agents which prevent the influx on neutrophils areable to increase the salvage of heart muscle cells.

Low concentrations of copolymers have little effect on plasma proteins.Higher concentrations, above the critical micelle concentration,activate complement via the alternate pathway. This provides furtherbenefit for treating heart attacks because the systemic activation ofcomplement causes the neutrophils to become unresponsive to complementchemotaxis. This prevents their migration into the heart tissue.

Continuing therapy is needed even after restoration of blood flow andsalvage of damaged tissue. The arteriosclerosis that caused the originalheart attack remains. American and European researchers have found thatarteriosclerosis still narrows the arteries in 70-80% of patients whoseclots were lysed by thrombolytic therapy. Many physicians believe thisobstruction must be opened for long term benefits. Balloon angioplastyis a procedure whereby a catheter with a small balloon is inserted intothe narrowed artery. The balloon is inflated, compresses theatherosclerotic plaque against the vessel wall and dilates the artery.The effectiveness of this procedure is limited by the effects ofischemia produced by the balloon, by embolization of atheromatousmaterial which lodges in distal vessels and by an increased tendency forimmediate or delayed thrombosis in the area damaged by the balloon. Theballoon tears the tissue exposing underlying collagen and lipidsubstances which induce formation of thrombi. The thrombus may occludethe vessel immediately or set up a sequence of events which leads toocclusion many days or weeks later. What is needed is a means ofrendering the surface of the dilated vessel less thrombogenic, improvingthe blood flow through the distal tissue and breaking the embolizedmaterial into smaller pieces which are less likely to produce embolicdamage.

Finally, lipid material on the atherosclerotic wall contributes to thebulk of the plaque which narrows the lumen of the artery and produces ahighly thrombogenic surface. What is needed is a method of extracting orcovering lipids from atherosclerotic plaques which leaves their surfacesless thrombogenic and reduces their bulk.

Use of copolymers prepared by the condensation of ethylene oxide andpropylene oxide to treat an embolus or a thrombus has been described(See U.S. Pat. No. 3,641,240). However, the effect was limited torecently formed, small (preferably microscopic) thrombi and emboli whichare composed primarily of platelets. The use of the ethylene oxide andpropylene oxide copolymer has little or no effect on a clot in a patientwho has suffered a severe coronary infarction. The clots that form inthese patients are large and stable clots. Stable clots are clots inwhich the fibrin that has formed from fibrinogen has undergone crosslinking. Fibrin which has undergone crosslinking is not effected bypresence of the ethylene oxide-propylene oxide copolymers. Thecopolymers only affect new clots in which the newly formed fibrin hasnot crosslinked.

Thus, a composition is needed that is capable of lysing a clot and, atthe same time, will prevent a second clot from reforming after theinitial clot has been cleared. Ideally, such a composition would alsoreduce as much as possible any damage that is caused by blockage ofblood supply to the tissue. Such a composition would thereby protect thepatient from any damage caused by the reformation of a clot. Inaddition, such a composition would be useful in removing clots fromsolid tumors, increasing flow through tortuous channels and therebyallow delivery of therapeutic drugs to the tumor.

SUMMARY OF THE INVENTION

In accordance with the present invention, a composition is provided thatis effective in dissolving blood clots and reestablishing andmaintaining blood flow through thrombosed coronary or other bloodvessels. The fibrinolytic composition of the present invention comprisesan enzyme, such as streptokinase, urokinase, prourokinase, tissueplasminogen activator, or other fibrinolytic enzyme, and asurface-active copolymer. The surface-active copolymer can be anethylene oxide-propylene oxide condensation product with the followinggeneral formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably about1750 to 3500, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes approximately 50% to 90% by weightof the compound.

The fibrinolytic composition of the present invention is usuallyadministered by intravenous injection into a patient

The present invention provides a composition that can be administered topatients who have a blood clot occluding a blood vessel The combinationof fibrinolytic enzyme and surface-active copolymer according to thepresent invention increases blood flow around a clot, rapidly lyses aclot, and provides further protection to the patient by preventing a newclot from forming and reducing reperfusion injury.

Another embodiment of the present invention is the combination of thesurface-active copolymer and oxygen radical scavengers such assuperoxide dismutase and mannitol. The present invention includes thecombination of surface-active copolymer, clot lysing enzyme and freeradical scavenger and also the combination of surface-active copolymerand free radical scavenger alone.

Because the fibrinolytic composition of the present invention stabilizesthe patient to a greater extent than treatments in the prior art, theadministration of more invasive procedures, such as balloon angioplasty,can be delayed thereby permitting selection of conditions for theinvasive treatment that are most favorable to the patient.

Another embodiment of the present invention is the use of thesurface-active copolymer to treat sickle cell disease. Yet anotherembodiment of the present invention is the use of the surface-activecopolymer to preserve organs for transplantation.

Accordingly it is an object of the present invention to provide acombination of fibrinolytic enzymes with a surface-active copolymer toproduce a synergistic action in lysing blood clots. This combination canbe formulated either with standard doses of enzyme to increase the rateor likelihood of lysing a clot or at lower doses of enzyme to reduceside effects while maintaining efficacy for lysing clots.

It is another object of the present invention to provide a compositionthat will reduce the need for anticoagulation in cardiac therapy andthereby lessen the danger of hemorrhage.

It is another object of the present invention to provide a compositionthat accelerates the dissolution of clots by freeing aggregatedplatelets and blocking further platelets from aggregating to the clot.

It is yet another object of the present invention to provide acomposition that can reduce the dose of fibrinolytic enzyme required tolyse a clot and thereby reduce the incidence of complications.

It is another object of the present invention to provide a compositionthat contains a surface-active copolymer and a free radical or oxygenscavenger, such as superoxide dismutase or mannitol.

It is a further object of the present invention to provide a compositionthat can promote blood flow through microvascular channels of tissuedamaged by ischemia and reduce the mount of tissue which undergoesnecrosis.

It is a further object of the present invention to provide a compositionthat will significantly reduce the risk of rethrombosis after treatmentwith fibrinolytic enzymes.

It is a further object of the present invention to provide a compositionthat will promote removal of lipids from atherosclerotic vessel wallsand thereby lessen the incidence of rethrombosis.

It is another object of the present invention to provide an improvedfibrinolytic composition that is capable of lysing fibrin depositsassociated with tumors.

It is another object of the present invention to provide a compositionwhich will increase blood flow through tortuous channels such as occurin tumors and during crisis of sickle cell disease.

It is another object of the present invention to provide an improvedcomposition and method for a vivo preservation of organs.

It is another object of the present invention to provide a compositionthat will reduce the risk of rethrombosis and thereby allow delay inadministering balloon angioplasty or other invasive procedures fortreatment of the compromised vessels.

It is another object of the present invention to provide a compositionwhich will reduce the risk of thrombosis immediately or at some timeafter invasive procedures such as balloon angioplasty which damageendothelial cells of the vasculature.

It is a further object of the present invention to provide a compositionto block the aggregation of platelets in blood vessels distal to thethrombosis and thereby limit extension of tissue damage.

It is yet another object of the present invention to provide acomposition to improve blood flow through and around tissue withextensive necrosis of myocardial or other cells thereby retardingnecrosis of additional myocardial tissue.

It is another object of the present invention to provide a compositionwhich will reduce the influx of neutrophils into damaged tissue andthereby reduce the extent of injury caused by toxic products ofneutrophils.

It is yet another object of the present invention to provide acomposition that will decrease the amount of ischemia caused blockage ofblood flow by a thrombus.

It is a further object of the present invention to provide a combinationof a thrombolytic enzyme, balloon angioplasty or other operativeprocedures and a surface-active copolymer to produce an improved methodof removing a thrombus or thrombogenic occlusion and reducingobstructive conditions which promote rethrombosis.

It is another object of the present invention to provide a compositionand method for the treatment of crisis in sickle cell

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows cumulative ¹²⁵ I fibrinogen (counts per minute) releasedfrom the columns plotted as a function of time.

DETAILED DESCRIPTION OF THE DISCLOSED EMBODIMENT

The present invention comprises a composition which provides asynergistic action in lysing blood clots and reestablishing andmaintaining blood flow through a thrombosed coronary vessel or otherblood vessel. The fibrinolytic composition of the present invention is asolution containing an effective concentration of a fibrinolytic enzymeand an effective concentration of a surface-active copolymer. Thecombination of the two components is effective in dissolving blood clotsthat are blocking blood vessels. In addition, the fibrinolyticcomposition of the present invention is highly effective in preventing ablood clot from reforming and in maintaining blood flow through theblood vessel and affected ischemic tissue.

The fibrinolytic composition of the present invention improves the flowof blood through narrow channels around clots and increases the deliveryof the fibrinolytic enzyme to the clot. The present invention alsospeeds the rate of dissolution of the clot by the enzyme and increasesthe proportion of clots lysed by promoting delivery of enzyme to clotswhich would not otherwise be exposed to sufficient enzyme for theirdissolution. In addition, the fibrinolytic composition of the presentinvention reduces the dose of enzyme required for particularapplications and thereby reduces the incidence of complications due toside effects caused by the enzymes.

The fibrinolytic composition of the present invention reduces the riskof immediate rethrombosis by accelerating the dissolution of clots,freeing aggregated platelets and blocking further platelets fromaggregating to the clot or clot site. By reducing the risk of immediaterethrombosis, the fibrinolytic composition of the present invention willallow delay of balloon angioplasty or other invasive procedures fortreatment of the compromised vessels which have become thrombosed. Thedelay will permit selection of conditions for invasive treatment mostfavorable to the patient

Solutions which may be employed in the preparation of the fibrinolyticcomposition of the present invention include, but are not limited to,saline (a solution of sodium chloride, containing 8.5 to 9.5 grams ofsodium chloride in 1000 cc of purified water), Ringer's solution,lactated Ringer's solution, Krebs-Ringer's solution, and various sugarsolutions. All of these solutions are well known to one of ordinaryskill in the art. However, it is to be understood that the fibrinolyticcomposition of the present invention may be administered as a solutionthat is not isotonic.

The surface-active copolymer is preferably an ethylene oxide-propyleneoxide condensation product with the following general formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably from1750 to 3500, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes from about 50% to 90% by weight ofthe compound. These copolymers are sold under the general trademark ofPluronic® polyols and are available from the BASF Corporation(Parsippany, N.J.).

The surface-active copolymers of the present invention are notmetabolized by the body and are quickly eliminated from the blood. Thehalf-life of the copolymer in the blood is approximately two hours. Itis to be understood that the surface-active copolymer in the improvedfibrinolytic composition of the present invention is not covalentlybound to any of the other components in the composition.

The polymer blocks are formed by condensation of ethylene oxide andpropylene oxide at elevated temperature and pressure in the presence ofa basic catalyst. There is some statistical variation in the number ofmonomer units which combine to form a polymer chain in each copolymer.The molecular weights given are approximations of the average weight ofcopolymer molecule in each preparation. It is to be understood that theblocks of propylene oxide and ethylene oxide do not have to be pure.Small amounts of other materials can be admixed so long as the overallphysical chemical properties am not substantially changed. A moredetailed discussion of the preparation of these products is found inU.S. Pat. No. 2,674,619 which is incorporated herein by reference.

Illustrative ethylene oxide-propylene oxide condensation products whichmay be employed in the preparation of the fibrinolytic composition ofthe present invention include, but are not limited to, the followingcopolymers:

1. A polyol with an average molecular weight of 4700 containingapproximately 80% by weight ethylene oxide.

2. A polyol with an avenge molecular weight of 3400 containingapproximately 50% by weight ethylene oxide.

3. A polyol with an avenge molecular weight of 7700 containingapproximately 70% by weight ethylene oxide.

4. A polyol with an avenge molecular weight of 14,600 containingapproximately 80% by weight ethylene oxide.

5. A polyol with an avenge molecular weight of 12,600 containingapproximately 70% by weight ethylene oxide.

6. A polyol with an avenge molecular weight of 9500 containingapproximately 90% by weight ethylene oxide.

The preferred ethylene oxide-propylene oxide copolymer for use in thefibrinolytic composition of the present invention is a copolymer havingthe following formula:

    HO((C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400.

The concentration of copolymer in the clot-dissolving composition of thepresent invention can vary depending the total volume of solution neededin the particular circumstances. The total amount of block copolymeremployed in the present invention will also vary depending on the sizeand type of thrombus or embolus, the particular copolymer employed, theparticular fibrinolytic enzyme employed, and the size and weight of thepatient.

The copolymer can be used over a wide range of concentrations with noadverse side effects. The copolymer is rapidly excreted intact; as muchas 90% of the copolymer administered is excreted in as little as threehours. Because of its low toxicity and the rapid clearance from thebody, the copolymer can be administered over a long period of time.

The fibrinolytic composition of the present invention may be employed byadmixing with blood in any standard manner. Preferably, however, thesolutions are intravenously injected into the blood stream either as abolus, slow drip or both. The solutions are generally admixed with theblood in a manner so as to maintain a substantially steady venouspressure.

It is to be understood that separate administration of a solution of thesurface-active copolymer and a fibrinolytic enzyme are contemplated inthe present invention. For example, a solution of the surface-activecopolymer and a solution of a fibrinolytic enzyme could be preparedseparately and administered simultaneously or sequentially to a patientsuffering from a thrombus blocking a coronary artery. Simultaneous orsequential administration of the two components (copolymer andfibrinolytic enzyme) of the fibrinolytic composition of the presentinvention has the same effect as administering the components togetherand is therefore contemplated in the present invention.

The fibrinolytic enzymes that can be used in the fibrinolyticcomposition of the present invention include, but are not limited to,streptokinase (available from Hoechst-Roussel under the WademarkStreptase®), urokinase (available from Abbot Laboratories, NorthChicago, Ill. under the trademark Abbokinase®) and tissue plasminogenactivator (Biopool AB, Umea, Sweden). The tissue plasminogen activatorcan be derived from eukaryotic cells such as human melanoma cells or canbe made by genetic engineering methods such as recombinant DNA. Some ofthe fibrinolytic enzymes are only sparingly soluble in water and musttherefore be emulsified with the surface-active copolymer beforeadministration to the patient.

Ideally, a bolus injection of the copolymer solution without the enzymeis administered before the present invention is administered. Forexample, a 3% solution of the copolymer in 5% isotonic dextrose isinjected within a two minute period so that the blood concentration ofcopolymer is approximately 0.6 mg/ml. In addition, it can beadvantageous to administer a solution of the copolymer by intravenousdrip at a rate of about 25 mg/kg body weight/hour to obtain of bloodconcentration of the copolymer of approximately 0.6 mg/ml for up to fourdays or longer following the administration of the fibrinolyticcomposition of the present invention. This treatment will aid inpreventing a clot from reforming.

Although the descriptions relate mostly to heart disease, it is to beunderstood that the fibrinolytic composition of the present invention isequally applicable to thrombosis in other parts of the body, such as thebrain, legs, lungs or gastrointestinal tract.

The present invention includes a method for dissolving clots in bloodvessels comprising the steps of injecting into a body a solution with aneffective concentration of a surface-active copolymer with the followingformula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of from 950 to 4000, preferably from 1750 to4000, and b is an integer such that the hydrophile portion representedby (C₂ H₄ O) constitutes from approximately 50% to 90% by weight of thecompound. The blood concentration of the surface active copolymer isbetween approximately 0.1 mg/ml and 6 mg/ml, preferably between 0.5mg/ml and 2 mg/ml. A solution with an effective concentration of afibrinolytic enzyme and an effective amount of the surface-activecopolymer is then injected into the body. After the clot is dissolved orlysed, a solution with an effective concentration of the surface-activecopolymer is then injected into the body. The concentration of thecopolymer is maintained between approximately 0.4 and 2 mg/ml of bloodfor between approximately 4 hours and 144 hours.

The present invention also includes a method of removing fibrin from atumor comprising the step of injecting into a body a solution with aclot dissolving composition, said composition comprising an effectiveamount of a fibrinolytic enzyme and an effective amount of asurface-active copolymer of the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably from1750 to 4000, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes from approximately 50% to 90% byweight of the compound. The fibrinolytic composition of the presentinvention can be administered directly to the minor.

Removal of the fibrin from the tumor increases the accessibility ofdiagnostic reagents to the tumor. The method also increases theaccessibility and sensitivity of the tumor to the chemotherapy.

The present invention includes a method of improving blood flow andoxygenation of a tumor comprising the step of injecting into a body asolution comprising an effective concentration of a surface-activecopolymer of the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably from1750 to 4000, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes from approximately 50% to 90% byweight of the compound.

The present invention also includes an improved ex vivo tissue perfusioncomposition comprising blood, a solution with an effective amount of ananticoagulant, and an effective amount of a surface-active copolymer ofthe following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably from1750 to 4000, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes from approximately 50% to 90% byweight of the compound. The ex vivo tissue perfusion composition can bediluted with a colloidal solution or a salt solution. The preferredanticoagulant is heparin. This embodiment of the present invention isuseful in prolonging the life of human or animal organs that are to beused in transplantation.

The present invention also includes a method of treating sickle celldisease comprising the step of injecting into a body a solution with afibrinolytic composition therein, the composition comprising aneffective concentration of a fibrinolytic enzyme and an effective amountof a surface-active copolymer of the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.2).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably from1750 to 4000, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes from approximately 50% to 90% byweight of the compound.

The present invention includes a method of treating sickle cell diseasecomprising the step of injecting into a body a solution comprising aneffective concentration of a surface-active copolymer of the followingformula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of approximately 950 to 4000, preferably from1750 to 4000, and b is an integer such that the hydrophile portionrepresented by (C₂ H₄ O) constitutes from approximately 50% to 90% byweight of the compound.

The following specific examples will illustrate the invention as itapplies in particular to dissolving clots in blood vessels and topreventing clots from reforming. It will be appreciated that otherexamples will be apparent to those of ordinary skill in the art and thatthe invention is not limited to these specific illustrative examples.

EXAMPLE I

Addition of the copolymer to a clot dissolving enzyme results in asynergistic effect on the clot dissolving activity of the enzyme asdemonstrated in this Example.

Sterile 1 ml tuberculin syringes are packed with 0.6 ml of 500 to 750micron glass beads (Polyscience, Inc., Warington, Pa.). The tips of thesyringes are plugged with nytex filters and a one-way plastic stopcock.Fresh frozen platelet-poor titrated plasma is spiked with 15 μCi/ml ¹²⁵I human fibrinogen (Sigma Chemical Co., St. Louis, Mo.). The radioactiveplasma is diluted 1:2 with normal saline, and recalcified with calcium(American Dade, Aquada, Puerto Rico) at 1 volume of calcium to 4 volumesdiluted plasma.

Radioactive fibrinogen is bound to the glass bead columns as follows:Equal volumes of the radioactively labelled recalcified plasma are addedto parallel bead columns and allowed to run through the beads with noexcess plasma above the beads. All procedures and manipulations of the"bead clots" are performed at 37° C. The bead/plasma clots are allowedto incubated for 30 minutes, then washed for 30 minutes with normalsaline. During the last 5 minutes of the wash with saline, the flowrates are monitored and columns whose flow rates are abnormally fast orslow are excluded from the experiment. Normal flow rates average 0.2ml/minute.

The copolymer that is used in this example has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The copolymer is prepared as a stock solution of 1%copolymer by weight in normal saline.

Blood containing t-PA, with or without the copolymer, is passed throughthe columns as follows: 10 ml of heparinized whole blood is drawn freshand mixed with t-PA (10 μg in 1 ml normal saline; single chain; SigmaChemical Co. St. Louis, Mo.). A volume of 5.5 ml of blood is mixed witheither 0.5 ml normal saline or 0.5 ml of copolymer stock solution. Onealiquot of a preparation of whole blood or whole blood diluted 1:3 withnormal saline is run on each column. Three ml of each blood preparationis added to a reservoir connected to each cola. Fractions are collectedevery minute until all flow ceased. The volume in each tube is measuredand radioactivity counted in a Tracor Analytic gamma counter(TmAnalytic, Inc., Elk Grove Village, Ill.) Appearance of radioactivityin the collection tubes indicates lysis of the clot.

The dam are summarized in Table A and FIG. 1. FIG. 1 shows cumulative¹²⁵ I fibrinogen (counts per minute) released from the columns plottedas a function of time.

                  TABLE A                                                         ______________________________________                                                                  Counts Counts                                                                              Counts                                         Time     Volume   Minute Minute                                                                              Minute                                 Perfusate*                                                                            (Minutes)                                                                              Recovered                                                                              (Volume)                                                                             (ml)  (Cumulative)                           ______________________________________                                        Blood, t-PA,                                                                          1        0.3      2031   6770  2031                                   Copolymer                                                                             2        0.25     3042   12168 5073                                           3        0.3      13051  43503 18124                                          4        0.2      40190  200950                                                                              58314                                          5        0.25     40260  161040                                                                              98574                                          6        0.25     40009  160036                                                                              138583                                 Blood, t-PA                                                                           1        0.15     885    5900  885                                            2        0.2      1330   6650  2215                                           3        0.2      3681   18405 5896                                           4        0.3      16333  54443 22229                                          5        0.4      24932  62330 47161                                          6        0.45     30545  67878 77706                                          7        0.6      40365  67275 118071                                 Blood,  2        0.8      340    425   340                                    Copolymer                                                                             3        0.7      351    501   691                                            4        0.6      270    450   961                                            5        0.6      226    377   1187                                           6        0.5      204    408   1391                                           7        0.4      178    445   1569                                   ______________________________________                                         *A simulated thrombus containing .sup.125 I fibrin is prepared as             described in the text. The ability of test preparations to dissolve the       fibrin is determined by measuring the rate of elution of radioactivity        from the column. The copolymer is not an enzyme and has no reactive           groups, so it is unable to lyse crosslinked fibrin, but it does increase      the fibrinolytic activity of tPA in this model which is designed to           simulate the structure and flow conditions of a coronary thrombus.       

As can be seen from Table A and FIG. 1, treatment of the radioactiveclot with the surface-active copolymer releases little of theradioactivity indicating no lysis of the clot. Administration of t-PA tothe clot causes release of radioactivity indicating lysis of the clot inthe column. However, when the surface-active copolymer is added to thesolution, the rate of lysis of the clot in the column is dramaticallyincreased. Thus, the combination of surface-active polymer and t-PAlysed the clot in the column at a significantly faster rate than didt-PA alone.

In other experiments, the model is modified by changing the size of thebeads, the concentration of plasma used to make the clot, the dilutionof blood or the concentration of enzyme or copolymer. In severalinstances, columns are produced in which whole blood fails to flow atall while blood with copolymer flows at a rate of about 0.05 ml/minute.t-PA in the blood is unable to dissolve any of the fibrin in such clotsas measured by release of ¹²⁵ I label because there is no flow of bloodthrough the clot. The use of copolymer with the blood and t-PA in suchsituations caused rapid lysis of the clot.

EXAMPLE II

The fibrinolytic composition is tested in an ex vivo rat heart model Thedetailed design of the system is described elsewhere. (See Paulson, etal., Basic Res. Cardiol., Vol. 81, pp. 180-187, 1986 which isincorporated herein by reference). This model measures the ability ofthe isolated heart to recover from a 30 to 90 minute ischemic periodwhere the flow of nutrients is reduced to 1 percent of normal orcompletely stopped, then followed by a 10 minute period of reperfusion.Three parameters measured: (1) cardiac output (CO); (2) left ventricularsystolic pressure (LVSP); and (3) left ventricular contraction (dp/dt).Assessment of heart recovery and mount of damage are discussed inPaulson, D. J. et al. Basic Res. Cardiol., Vol. 79, pp. 551-561, 1984.

In this experiment, hearts are perfused with washed whole human bloodwith no heparin added. Flow is completely stopped for 30 minutes,followed by 10 minutes reperfusion with washed whole human blood withoutheparin but with the additive or additives indicated in Table B. Thecopolymer that is used in this example has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The copolymer is prepared as a stock solution of 1%copolymer by weight in normal saline.

The results of the test are as follows. The final concentration of thesurface-active copolymer used in this Example is 0.68 mg/ml. Thestreptokinase that is used in this Example can be obtained from SigmaChemical Company, St. Louis, Mo. Streptokinase is administered at aconcentration of 100 units/hem. The results are shown in Table B.

                  TABLE B                                                         ______________________________________                                                   Percent Cardiac Recovery                                                      (Values are mean)                                                  Additions    CO          LVSP    dp/dt                                        ______________________________________                                        Whole Blood   5          24      10                                           with Copolymer                                                                             38          82      65                                           with Streptokinase                                                                         33          75      60                                           with Copolymer and                                                                          58*        88      78                                           Streptokinase                                                                 ______________________________________                                         *p < 0.05 for cardiac output (CO) differences between the combination of      Copolymer and Streptokinase and (a) whole blood ischemic control, (b)         Copolymer only, and (c) Streptokinase only. Student's t test was used to      determine differences between independent means. A result of p < 0.05 was     regarded as significant.                                                 

As can be seen from Table B, the copolymer and streptokinase combinationclearly protected the heart better than the copolymer or streptokinasealone.

EXAMPLE III

For treating a patient weighing about 180 lbs with a pulmonary embolism,reconstitute 500 mg of urokinase (Abbokinase, Abbot Laboratories, NorthChicago, Ill.) in 105 ml of sterile water. To the urokinase solution add90 ml of an 0.9% sodium chloride solution containing 6 grams of anethylene oxide-propylene oxide copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The urokinase and the copolymer are thoroughly mixedto form a homogeneous solution. The final volume of the solution is 195ml.

Administer the fibrinolytic composition of the present invention bymeans of a constant infusion pump that is capable of delivering a totalvolume of 195 ml. A priming dose of the fibrinolytic composition of thepresent invention is administered at a rate of 90 ml/hour over a periodof 10 minutes. This is followed by a continuous infusion of the presentinvention at a rate of 15 ml/hour for 12 hours. Because some of thefibrinolytic composition of the present invention will remain in thetubing at the end of an infusion pump delivery cycle, the remainingsolution is flushed out of the tube by administering a solution of 0.9%sodium chloride at a rate of 15 ml/hour.

EXAMPLE IV

For treating a patient with a coronary artery thrombi, reconstitute 75mg of urokinase (Abbokinase, Abbot Laboratories, North Chicago, Ill.) in15.6 ml of sterile water. To the urokinase solution add 300 ml of 5%dextrose solution containing 15 grams of an ethylene oxide-propyleneoxide copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The urokinase and the copolymer are thoroughly mixedto form a homogeneous solution. The solution is then diluted with 5%dextrose to a final volume of 500 ml.

The solution comprising the present invention is infused into theoccluded artery at a rate of 4 ml per minute for periods up to 2 hours.To determine response to the solution of the fibrinolytic composition ofthe present invention, periodic angiography is performed.

EXAMPLE V

For treating a patient weighing about 180 lbs with a pulmonary embolism,reconstitute 500 mg of urokinase (Abbokinase, Abbot Laboratories, NorthChicago, Ill.) in 105 ml of sterile water. To the urokinase solution add90 ml of an 0.9% sodium chloride solution containing (5.0 grams of anethylene oxide-propylene oxide copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound isapproximately 8400. The urokinase and the copolymer are thoroughly mixedto form a homogeneous solution. The solution is then diluted with 0.9%sodium chloride to a final volume of 195 ml.

Administer 137 ml of a 5% isotonic dextrose solution with 3% wt/volethylene oxide-propylene oxide copolymer lysed therein to the patientover a 2 minute period. This gives a blood concentration of copolymer ofapproximately 0.6 mg/ml (assuming blood is 8% of body weight)

The fibrinolytic composition of the present invention is thenimmediately administered by means of a constant infusion pump that iscapable of delivering a total volume of 195 ml. A priming dose of thepresent invention is administered at a rate of 90 ml/hour over a periodof 10 minutes. This is followed by a continuous infusion of the presentinvention at a rate of 15 ml/hour for 12 hours. Because some of thepresent invention will remain in the tubing at the end of an infusionpump delivery cycle, the remaining solution is flushed out of the tubeby administering a solution of 0.9% sodium chloride containing 3.0%copolymer at a rate of 15 ml/hour.

After the clot is lysed, a solution of the copolymer is administered byintravenous drip at a rate of about 25 mg/kg body weight/hour tomaintain a blood concentration of the copolymer of approximately 0.6mg/ml. The administration of the copolymer solution is continued forfour days following the administration of the fibrinolytic compositionof the present invention.

EXAMPLE VI

For ex vivo organ preservation in preparation for transplantation,reconstitute 1000 units of heparin (Sigma Chemical Company, St. Louis,Mo.) in 200 ml of normal (0.9%) sodium chloride solution and add 1.36gof the copolymer of the present invention and resuspend washed wholehuman blood to formulate the perfusion medium. The copolymer has thefollowing formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

Hearts excised from anesthetized Sprague-Dawley rats were perfused for10 minutes with (a) blood and heparin or with (b) blood, heparin andcopolymer following a 90 minute low-flow ischemia. Cardiac output (CO),left ventricular systolic pressure (LVSP) and left ventricularcontraction (dp/dt) were determined and are expressed as percent ofrecovery as compared to normal hearts. Ischemic animals' hearts whichreceived blood with heparin showed poor recovery: 12% CO, 44% LVSP and34% dp/dt. Hearts given blood, heparin and copolymer showed excellentrecovery: 90% CO, 92% LVSP, and 84% dp/dt. For the heparin withcopolymer group, all three parameters were statistically different(p<0.01) as compared to the ischemic control group (heparin only).Differences between independent means were determined by the Student's ttest.

EXAMPLE VII

A test is performed to demonstrate the ability of the combination ofsuperoxide dismutase and an appropriate copolymer to produce greaterprotection of ischemic myocardium from reperfusion injury associatedwith oxygen radicals and other factors than superoxide dismutase alone.

Under general anesthesia (sodium thiopental 25 mg/kg), the animals areintubated and ventilated with 70% oxygen at a rate of 12 breaths perminute. A satisfactory level of anesthesia is maintained withintermittent boluses of pentothal as required. After skin preparation, aleft anterior thoracotomy is performed, the pericardium incised and theheart exposed. The left anterior descending coronary artery isidentified, isolated and encircled with a snare 1 cm from its origin.Temporary left anterior descending coronary artery occlusion isaccomplished by tightening the snare and continues for 90 minutes.During the procedure, the heart rate and blood pressure are monitoredutilizing a Hewlett-Packard 7758B 8-channel recorder. Arterial bloodpressure is monitored through an 18 gauge indwelling catheter in theright femoral artery and measured with a Hewlett-Packard quartztransducer. Electrocardiographic evidence for anteroseptal myocardialischemia is also monitored. Reperfusion of the ligated vessel after 90minutes of ischemia is achieved by a gradual release of the snare toprevent the hyperemic response. A defibrillator is available in the roomas are all necessary cardiotonic drugs in the event of cardiacfibrillation or circulatory collapse due to the left anterior descendingcoronary artery ligation. Therapeutic agents are infused in conjunctionwith reperfusion as follows: bovine superoxide dismutase withapproximately 3000 units of activity per milligram assayed by the methodof McCord, J. Biol. Chem., Vol. 244, p. 6049 (1969) is obtained fromSigma Chemical Company, St. Louis, Mo. It is dissolved in 100 ml ofnormal saline and infused intravenously over 90 minutes starting 15minutes before restoration of perfusion. This simulates the effectswhich occur during lysis of a coronary thrombus.

A solution of copolymer is prepared at 2% weight/volume in saline. It isgiven intravenously as a bolus over 2 minutes in a dose sufficient toachieve a blood level of 0.6 mg/ml followed by a constant infusion ofapproximately 25 mg/kg/hour in order to maintain the blood level ofapproximately 0.6 mg/ml for the remainder of the experiment.

The ethylene oxide-propylene oxide copolymer has the following generalformula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 and the total molecular weight of the compound is8400.

The synergistic effect of the combination is demonstrated by comparingthe results of dogs treated with both the copolymer and superoxidedismutase with those treated with either material alone or no treatment.

Agents are infused intravenously utilizing an IVAC 560 infusion pump.Infusion begins 15 minutes prior to release of the mare and continuesuntil the total dose for each group has been altered. The chest isclosed in layers. A chest tube is utilized to evacuate the pneumothoraxand is removed when spontaneous respirations resume. Intravenous fluidsare given (Lactated Ringer's Solution) to compensate for the 24 hour NPOperiod preceding the operation, in addition to a 3 to 1 ratio tocompensate for blood loss. The animals are then maintained and followedclosely for the next 24 hours. Each animal is then returned to theoperating suite and under general anesthesia the previous incision isreopened. The animal is sacrificed utilizing a barbiturate overdose. Theheart and proximal 4 cm of ascending aorta is excised being Sure toinclude the origins of the coronary arteries.

All groups are subjected to the same procedures for identification ofthe area of the myocardium at risk for infarction and the area actuallyinfarcted.

This technique involves perfusion of the left anterior descendingcoronary artery with 2,3,5-triphenyltetrazolium chloride, which stainsthe intact myocardium red and leaves the infarcted myocardium unstained.The limits of the area of myocardium at risk are determined by perfusingthe remainder of the coronary system, via the aortic root, with EvansBlue dye. The area at risk is defined by a lack of Evans Blue stain.

It should be understood, of course, that the foregoing relates only to apreferred embodiment of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

We claim:
 1. A method of treating tumors in a human or animal comprisingthe step of administering to the human or animal with the tumor acomposition comprising a chemotherapeutic agent and a solutioncomprising an effective concentration of a surface-active copolymer withthe following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 950 to 4000 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 2. The method of claim 1, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 1200 to 3500 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 3. The method of claim 1, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 Daltons and the total molecular weight of thecompound is approximately 8400 Daltons.
 4. A method of treating tumorsin a human or animal comprising the step of intravenously administeringto the human or animal with the tumor a composition comprising achemotherapeutic agent and a solution comprising an effectiveconcentration of a surface-active copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 950 to 4000 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 5. The method of claim 4, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 1200 to 3500 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 6. The method of claim 4, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 Daltons and the total molecular weight of thecompound is approximately 8400 Daltons.
 7. The method of claim 4,wherein the chemotherapeutic agent and the surface active copolymer areadministered separately.
 8. A method of treating tumors in a human oranimal comprising the step of intramuscularly administering to the humanor animal with the tumor a composition comprising a chemotherapeuticagent and a solution comprising an effective concentration of asurface-active copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 950 to 4000 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 9. The method of claim 8, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 1200 to 3500 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 10. The method of claim 8 wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 Daltons and the total molecular weight of thecompound is approximately 8400 Daltons.
 11. The method of claim 8,wherein the chemotherapeutic agent and the surface active copolymer areadministered separately.
 12. A method of treating tumors in a human oranimal comprising the step of administering to the human or animal withthe tumor a composition comprising a chemotherapeutic agent and anon-emulsion, homogenous solution comprising an effective concentrationof a surface-active copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 950 to 4000 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 13. The method of claim 12, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 1200 to 3500 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 14. The method of claim 12, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 Daltons and the total molecular weight of thecompound is approximately 8400 Daltons.
 15. The method of claim 12,wherein the chemotherapeutic agent and the surface active copolymer areadministered separately.
 16. A method of treating tumors in a human oranimal comprising the step of intravenously administering to the humanor animal with the tumor a composition comprising a chemotherapeuticagent and a non-emulsion, homogenous solution comprising an effectiveconcentration of a surface-active copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 950 to 4000 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 17. The method of claim 16, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 1200 to 3500 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 18. The method of claim 16, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 Daltons and the total molecular weight of thecompound is approximately 8400 Daltons.
 19. The method of claim 16,wherein the chemotherapeutic agent and the surface active copolymer areadministered separately.
 20. A method of treating tumors in a human oranimal comprising the step of intramuscularly administering to the humanor animal with the tumor a composition comprising a chemotherapeuticagent and a non-emulsion, homogenous solution comprising an effectiveconcentration of a surface-active copolymer with the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 950 to 4000 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 21. The method of claim 20, wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein a is an integer such that the hydrophobe represented by (C₃ H₆O) has a molecular weight of between approximately 1200 to 3500 Daltons,and b is an integer such that the hydrophile portion represented by (C₂H₄ O) constitutes between approximately 50% to 90% by weight of thecopolymer.
 22. The method of claim 20 wherein the surface-activecopolymer has the following formula:

    HO(C.sub.2 H.sub.4 O).sub.b (C.sub.3 H.sub.6 O).sub.a (C.sub.2 H.sub.4 O).sub.b H

wherein the molecular weight of the hydrophobe (C₃ H₆ O) isapproximately 1750 Daltons and the total molecular weight of thecompound is approximately 8400 Daltons.
 23. The method of claim 20,wherein the chemotherapeutic agent and the surface active copolymer areadministered separately.